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DETERMINATION AND PRECONCENTRATION OF MANGANESE

USING IONIC LIQUID BASED MICROEXTRACTION TECHNIQUE IN
BIOLOGICAL SAMPLES
Fariba Tadayon1, Mahnoosh Hanasaei2, Masoumeh Madadi3
Department of Chemistry,
North Tehran Branch, Islamic Azad University, Tehran,
IRAN.
1

F_tadayon@iau-tnb.ac.ir

ABSTRACT
In this work, a procedure for preconcentration and determination of manganese
using modified cold-induced aggregation microextraction(CIAME) based ionic
liquid with the reagent 1-(2-pyridylazo)-2-naphthol (PAN) as complexing reagent
was developed. In the preconcentration step, the mixture was cooled in an ice bath
and manganese from 10mL of an aqueous solution was extracted into70L of ionic
liquid, 1-hexyl-3-methylimidazolium hexafluorophosphate [Hmim][PF6]. After
centrifuging the extraction phase was settled. Then, the absorbance of the complex
was measured by UV-Vis spectrophotometry. In this study, the effect of matrix ions
on the extraction efficiency of Mn +2were investigated. Also the optimization
extraction conditions, such as: pH, PAN concentration, extraction time, addition of
salt, ionic liquid volume and centrifugation rate were studied. The relative standard
deviation was 1.14 % (n=4). The detection limit of 0.54 ngL -1 and enrichment factor
of 70 for Mn+2 were achieved. The presented procedure was successfully applied for
determination of manganese in environmental and biological samples.
Keywords: Cold-induced aggregation microextraction Ionic Liquid, Manganese,
UV-Vis spectrophotometry

INTRODUCTION
Manganese has been called as a rare mineral. Its found in trace amounts in human body and
plays a role in establishing tissue fat and cholesterol, bone, blood clotting factors and proteins.
Manganese is a component of the composition of superoxide dismutase (Critchfield et al., 1993,
Kazi et al., 2008). Therefore, it is important from an analytical point of view to develop
sensitive methods for determination of Mn +2 (Kazi et al., 2009). Different techniques have been
used for measuring of manganese such as: flame atomic absorption spectrometry (FAAS) (Arpa
et al., 2010), electro thermal atomic absorption spectrometry (ETAAS) ( Sarzanini et al., 2001.,
Tokman, 2007) and inductively coupled plasma-optical emission spectrometry (ICP-OES)
(Ranjbar et al., 2012). Also, UV-Vis spectrophotometry is a significant instrument on the
determination of trace elements in various samples due to apart from the high sensitivity and
precision, its simple, inexpensive and available compared to other devices. The direct
measurement of manganese by UV-Vis spectrophotometry is associated with the interference
matrix, because the Mn+2 concentrations are low in environmental and biological samples. The
most effective way to avoid this problem is removal of interferences and increasing the
concentration of the analyte. Therefore is requiring a separation/preconcentration step before the
measurement (Vaezzadeh et al., 2010). Microextraction techniques are very efficient for the
preconcentration of metal ions at trace levels in other samples (Yousefi and Shemirani, 2010).
Several microextraction techniques have been suggested for the preconcentration of manganese
that can be pointed to: single drop microextraction (SDME) (Manzoori et al., 2009), membrane
solid phase
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microextraction (MSPME) (Cui et al., 2011), dispersive liquidliquid microextraction (DLLME)

(Balogh et al., 2012). In various reports have been exhibited to the usage of ionic liquids (ILs)
on preconcentration of trace metal ions (Violina et al., 2006). Ionic liquids have unique physical
and chemical properties such as: negligible vapor pressure, non-flammable, good thermal
stability, non-volatility, adjustable viscosity and a worthy replacement for organic and toxic
solvents (Li et al., 2009, Martinis et al., 2009, Molaakbari et al., 2011).

In this work, 1-hexyl-3-methylimidazolium hexaflourophosphate [Hmim][PF 6] based coldinduced aggregation microextraction( CIAME) was applied for the determination of
manganese in water and biological samples. CIAME method is easy, simple and
inexpensive for preconcentration of metal ions, so it can easily settle up to 40% salt
(Mahpishanian and Shemirani, 2010). CIAME consist of three simple steps: (1)
solubilization of the analyte in the ion liquid phase; (2) clouding; (3) phases separation for
analysis (Baghdadi and Shemirani, 2008, 2009). In this method manganese was initially
complexed with1-(2-pyridylazo)-2-naphthol (PAN) reagent, so the complex is extracted
into the IL- phase and is measured by UV-Vis spectrophotometry.
EXPERIMENTAL
Apparatus
A lambda 25 UV-Vis spectrometer was purchased from Perkin-Elmer (USA) and used
recording the spectra. A Kokusan Japan centrifuge (H-11 n) was used to accelerate the
phase separation process and a Jeio Tech BW-05G water bath was obtained from Hettich.
A100L microsyringe (Hamilton) was employed to inject ionic liquid extracting phase to
the sample solution.
Reagents and Materials
All of the reagents used were of analytical grade. PAN, ethanol, ammonium hydroxide,
ammonium chloride, acetonitrile and all salts used, were obtained from E.Merck and
[Hmim][PF6] was purchased from Aldrich.
-1

+2

A1000 mgL Mn stock standard solution was prepared from appropriate amounts of
manganese nitrate salt(E.Merck) in deionized water, according to the standard
method
and
-3
-1
diluted daily for obtaining work solutions prior to use. A solution of 110 molL PAN
was prepared
by dissolving appropriate amount of this reagent in pure ethanol and also 0.85
-1
mgL of [Hmim][PF6] was prepared in acetonitrile. A buffer solution
(pH 9.5) was
-1
prepared by mixing of 50mL ammonium
hydroxide
solution
(1
molL
)
and 50mL of
-1
ammonium chloride solution (0.18 molL ).
Modified CIAME Procedure
+2

-5

-1

The pH of 10 mL of standard solution or sample containing Mn and PAN (110 molL ) in the
screw-cap conical-bottom glass centrifuge tube was adjusted
to 9.5 using ammonium hydroxide/
-1
ammonium chloride. Then, 70L [Hmim][PF 6] 0.85 mgL was injected into the tube, and was
protected in a thermostated bath at 50 C for 4 min. Then it was placed for 15 min in an ice bath
until extraction was occurred. Afterwards, the mixture was centrifuged for 5min at 4000 rpm.
Therefore, the fine droplets of IL inhabited at the bottom of the centrifuge tube. Bulk aqueous phase
was removed simply by pipette. So IL-phase was dissolved in100L of ethanol solution (85%) and
transferred to quartz cell. The absorbance of the complex was measured at 549nm.

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Real Samples
Cow tail and human Hair samples
Hair samples were washed with 1% (w/v) (DDTC), 0.1M HCl and deionized water.
Afterwards, the hair samples dried in oven at 70C for 8 hours and then digested the next
day (Tadayon et al, 2012). 1g of washed hair samples were weighted and transferred to a
Teflon bombs and 10mL of concentrated HNO 3 (65%) was added. The samples were heated
on a plate about 100C for 2h.After dissolution, the solution was allowed to cool and 5mL
of H2O2 (30%) was added. The mixture was heated at70C for 2h(S-Tehrani and Tadayon,
2009). After digestion, the sample was diluted with deionized water and was treated
according to the given procedure. Mn +2 was measured in the real samples prepared with
UV-Vis spectrophotometry and ETAAS in order to compare.
Tap Water
The sample was prepared of drinking water in Tehran city. Mn +2 was measured using of
spiked method in this sample.
RESULTS AND DISCUSSION
Effect of pH
pH plays an important role in the extraction efficiency of complex . The effect of pH on the
extraction of manganese was studied by varying the pH within the ranges 4-12. As shown
in Figure.1, absorbance increased in the range 4-9.5 of pH and then started to decrease.
Because PAN activity decreases in the acidic qualification due to protonation of oxygen and
nitrogen. Also metal settle up the form of hydroxide in strongly alkaline perimeter.
Therefore, pH 9.5 was selected as the best pH.
0.15

Absorbance

0.12
0.09
0.06
0.03
0
0

6
pH

10

12

Figure 1.Effect of pH on manganese extraction: sample volume, 10mL; manganese concentration, 54

-5

ngL-1; PAN concentration, 10 M; IL volume, 65L; diluting agent, 100L.

Effect of PAN
PAN is a azo compound which act as a ligand. In order to study the influence of PAN
concentration on
the absorbance of complex, different concentrations of the PAN in the
range of 510-6-1.310-5 molL-1 were studied. The results illustrated in Fig.2. As can be
-5
seen, -1extraction efficiency increased with increasing ligand concentration-5 up to110
-1
molL and then remains constant. Therefore, PANconcentration of 110 molL was
selected for subsequent experiments.
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0.15
Absorbance

0.12
0.09
0.06
0.03
0
0

10

12

14

Concentration of PAN(molL-1)10-6
Figure 2. Effect of concentration
PAN on manganese extraction: sample volume, 10mL; manganese
concentration, 54 ngL-1; pH 9.5; IL volume, 65 L; diluting agent, 100L

Effect of NaNO3
In order inquire into the influence of the ionic strength on the complex absorbtion, several
experiments were performed with distinctive NaNO 3 concentrations (10-60% WV -1). NaNO3
salt in water is only the ionic form and adding value to salt is increased the ionic strength
sample solution. But if too much salt is added, displacement of PF 6 ion with NO3 ion occurs
and cause the [Hmim][NO3] salt that is completely soluble in water. This process reduces
extraction efficiency. A concentration of 40% NaNO3 (W V-1) was selected for this work.
Effect of [Hmim][PF6]and Centrifuge Condition

Extraction efficiency of complex can be remarkably affected by the ion liquid amount.
Thus, the IL volume is one of the most important parameters in development of
preconcentration method, because it determines the sensitivity intensification of the
method. The effect of IL volume was studied in range (20-100L). 70L was chosen as the
optimum volume. The results are depicted in Fig.3.

Absorbance

The final extraction performance is related to the complete separation of the ionic liquid.
Thus centrifugation is necessary step to obtain two discrete phases. In order to complete
sedimentation of IL phase, different rates of centrifugation (1000-6000 rpm) were studied
and the rate of 4000 rpm was selected as the best spot.
0.15
0.12
0.09
0.06
0.03
0
0

20

40

60

80

100

120

IL volume(L)
Figure 3. Effect of ionic
liquid volume on manganese extraction: sample volume, 10mL; manganese
concentration, 54 ngL-1; pH 9.5; PAN concentration, 110-5 M; diluting agent, 100 L.
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Effect of Extraction Time and Temperature

Extraction time is one of the important experimental parameters in more analytical
processes, particularly in microextraction methods. In M-CIAME based IL, the extraction
time is defined as the interval time between finishing the disruption of [Hmim][PF 6] and
starting to centrifuge. Hence, extraction time plays an important role in this new procedure.
Various times were studied to extracting in the range of 5-30min (Fig.4). The results explain
that in 15 min, contact between extraction solvent and aqueous phase is very high.
Temperature is necessary driving force for the ionic liquid distribution and complex
stability. Before shaking a solution including IL, they were heated the range of 20-50C. At
high temperature 50C could create permanence problems for chelating agent. Therefore
50C was selected as the most suitable temperature.
0.15
Absorbance

0.12
0.09
0.06
0.03
0
0

10

20
Extraction Time (min)

30

40

Figure 4. Effect of extraction time on manganese extraction: sample volume, 10mL;manganese

concentration, 54 ngL-1; pH 9.5; PAN concentration, 110 -5 M; IL volume, 70L; diluting agent,
100L.

Interferences
+2

The effect of foreign ions in cold-induced aggregation microextraction of Mn using PAN

were studied in the presence of several amounts of other ions under the optimized
conditions. The tolerance limit for interference of each kind was set at 5% in the analytical
signal obtained for a solution including Mn +2, except for Ni+2 and Pb+2.
Table1. Effect of foreign ions the recovery of manganese (54 ngL-1)
Foreign Ion
Cr3+
Ni2+

Mg2+
Co2+

Cu2+
Na1+
K1+

Pb2+
Fe3+

Hg2+
ClN
S

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Molar ratio(Ion/Mn+2)
1
1
100
1
10
1000
100
1
10
100
100
100
100
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Mn recovery (%)
90.37
87.1
92.59
101.4
100
97.77
99.25
107
94.07
99.25
99.25
97.77
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But because of real samples are diluted 2 times and the amount of nickel and lead in these
samples is very low, were found not interference from the coexisting ions for the
+2
determination of Mn . The maximum tolerances of the examined foreign ions are given in
Table 1.
Determination of Manganese in Real Samples
The M-CIAME method was applied for the determination of manganese with UV-Vis
spectrophotometry and electrothermal atomic absorption spectrometry (ETAAS) in order to
compare in environmental and biological samples including (tap water, human and cow
hairs). The results are shown in Table 2, as it can be seen the results obtained by UV-Vis
spectrophotometry they were in good agreement with those obtained by the ETAAS.
Table 2.1 Determination of Mn+2 in human and cow hairs (n=4)
UV-Vis (Founded Mn+2 mean SDa)

Sample
Human hair(gg-1)
Cow tail hair( gg-1)

ET-AAS

0.51 0.075
0.33 0.051

0.52 0.03
0.350.036

Table 2.2 Determination of Mn+2 inTap water(n=4)

Sample

Added

Tap waterb (ngL-1)

UV-Vis
(Founded Mn+2 mean SDa )
0. 540.02

Recovery (%)

54.71
55.23 0.052
Standard deviation(n=3)
b
From drinking water system of Tehran, Iran

ET-AAS

99.96

55.020.041

The Analytical Figures of Merit and Comparison of M-CIAME with Other Methods
According to the given procedure, the calibration graph was constructed by processing
standard solutions of manganese under the optimum condition. The linear concentration
-1
range was 0.054-540 gL with correlation coefficient of 0.997. The precision of the
method was calculated as the relative standard deviation of 4 independent measurements
-1
for 54 ngL manganese. Likewise, the enrichment factor (EF) is defined as the ratio of the
calibration curve slope for manganese before and after the preconcentration step. The limit
of detection, calculated based 3S b/m (where Sb is standard deviation of the blank signal and
m is the slope of calibration curve after extraction).The results are summarized in Table 3.
Table 3. Analytical characteristics of modified CLAME method
Mn+2 with preconcentration

Parameter
Correlation coefficient(R2)
Limit of detection(LOD)

0. 549 ng L-1

Enrichment factor(EF)

70

%RSD(n=4)

1.14%(54ng L-1)

0.997

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The proposed method was compared with the other preconcentration methods used for the
determination of manganese and the results are shown in Table 4. All these results indicate
that in comparison with other reported methods, the M-CIAME method has low LOD and
good enhancement factor. Also is a rapid, environmentally friendly and simple technique
that can be used for preconcentration of metal ions in different samples. In addition, IL was
used instead of volatile and toxic organic solvent as the extraction phase.
Table 4. Comparison of modified CIAME with other methods for determination of manganese
(II)
LOD

Method
SDME(ETAAS)

MSPME(ICP-OES)
CPE(FAAS)

U-AEME(GFAAS)
e

DLLME (UV-Vis
spectrophotometric)
a

RSD

( gL )

(%)

0.024

5.5

Mn(II)
Concentration
-1
( gL )
0.5

0.61

6.2

0.01

environmental

Cui et al, 2011

2.9

1.3

200

water samples
Water and

Arpa et al, 2010

0.3

3.3

food samples
water samples

Mohadesi and Falahnejad,

1.2

55

water samples

2012
Balogh et al, 2012

-1

Sample

Reference

water samples

Manzoori et al, 2009

Single drop microextraction(electrothermal atomic absorption spectrometry)

Membrane solid phase microextraction ( inductively coupled plasma-optical emission

spectrometry)
c
Cloud point extraction (flame atomic absorption spectrometry)
d

Ultrasound-assisted emulsification microextraction( graphite furnace atomic absorption

spectrometry)
e
dispersive liquidliquid microextraction
CONCLUSION
In this work, a novel method of cold-induced aggregation microextraction based ionic
liquid was demonstrated for the preconcentration and determination of manganese in water
and biological samples.
This technique acts as a selective and can be separated the analyte from interferences. Also
it is simple, rapid, cheap, environment-friendly and can easily settle up to 40% salt. The use
of ionic liquid (green solvent) instead of toxic and organic solvents is considered of the
important characteristics of this technique. In this study good accuracy and precisions, very
low detection limit was achieved for manganese. Also results obtained of this method were
checked by ET-AAS spectrometry, and observed the results completely agreement with
each other.
ACKNOWLEDGMENT
With special thanks of Ms.Vahideh Mohajeri and Ms. Mahtab Rezazadeh that supported us
in this project.

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